CN106146630B - A kind of mould albumen exciton EPTP and its application in raising disease resistance of plant - Google Patents

Abstract

A kind of mould albumen exciton EPTP and its application in raising disease resistance of plant, the protein sequence that the present invention provides EPTP albumen exciton is shown in SEQ ID NO.2, and molecular size range is 19247 Da.The phenylalanine lyase of EPTP evoking tobacco blade, peroxidase, polyphenol oxidase activity dramatically increase, while during the exciton evoking tobacco allergic reaction, allergic reaction marker genehin1It is induced to express；Semiquatitative RT-PCR assay the result shows that, EPTP can obtain systemic acquired resistance with evoking tobacco, cause tobacco leaf resistant gene transcriptional level to raise, and induce the expression of salicylate pathway and jasmonic approach related resistance genes.Plant can be improved after EPTP application to virus, the resistance of pathogen, act on efficient, no pollution to the environment, chemical pesticide usage amount can be reduced, had broad application prospects as plant immune activator.

Description

A kind of mould albumen exciton EPTP and its application in raising disease resistance of plant

Technical field

The invention belongs to agromicrobiology technology and Biochemistry and Molecular Biology technical fields, and in particular to a kind of Mould albumen exciton EPTP further relates to it and applies in improving disease resistance of plant.

Background technique

Albumen exciton can induce the resistance of plant, enhance plant to the defence capability of a variety of pathogens, and do not generate poison Side effect and the resistance feature for not causing pathogen, effect in biocontrol of plant disease more and more attention has been paid to.At present Albumen exciton has shown preferable application prospect on inducing plant resistance, however reported albumen exciton multi-source In bacterium hapin albumen, the albumen exciton discovery derived from fungi is less.

Fungi has the resource of antiviral active substance very abundant, and protein and peptide is wherein important one kind.Research It proves, albumen exciton encoding gene is important new gene resource, the excavation and transgenosis of binding protein exciton new gene Plant function research is beneficial to the developing of biological control new way and cultivates excellent New Crop Varieties (Tang Hongkun etc. 2010).The report about mycoprotein exciton has at present, and researcher obstructs spore bacterium (Monilinia fructicola) from clump Isolated different albumen exciton (Cui Yanling 2003) in mycelium, fermentation liquid or cell wall；Phytophthora The Elicitin of (Phytophthora sp.) secretion can make tobacco that allergic reaction occur and induce generation system resistance (Kamoun et al 2001), the allergic reaction (Chen 1991 of activated plant that can be strong；Du 1997)；From Alternariaspp, The New Type Activator Protein (Guo Guangjun etc. 2003) isolated and purified in the fungies such as Trichoderma and sickle-like bacteria, activator protein can pass through The metabolic regulation of plant is excited, via plant immunity is enhanced, further increase the content of plant chlorophyll and promotes root, stem and leaf Growth improves crop yield (Tang Hongkun etc. 2010；2005) Sunbo is glad to be waited；From Fusarium oxysporum (Fusarium oxysporum) Isolated Nep1-like proteins (NLPs) albumen (Gijzen 2006), research shows that NLPs albumen is in lower concentration The lower activity that plant defense enzyme can be improved.In a variety of dicotyledons, NLPs is capable of the resistance correlated response of activated plant, It such as forms protective plant protecting agent, generate Ethylene Signal molecule, up-regulated expression resistance related gene and cell death (Fellbrich 2002)。

Albumen exciton can excite plant that defense reaction occurs, and the main result to the raw resistance of plant inducer artificial delivery includes: Cause the allergic reaction and induction plant generation system resistance of plant.Allergic reaction be plant resist pathogen reaction it One, transmitted by signaling molecule, programmed death related gene pathogen infection position cell express, cause the cell and Peripheral cell is dead, so that pathogen, which is trapped in non-viable non-apoptotic cell and then reduces it, endangers (Lindgren et al 1997)；Together When, the plant parts not infected also have relevant resistance, this may be caused by the expression of related gene, or due to The transfer and transport of resisting substance cause.These processes require mediation (the Zhao et of signaling molecule participation and signal path al 2005)。

The virosis of China crops occurs seriously, to cause huge economic loss, so solving crop virosis Problem is most important.Due to becoming increasingly conspicuous for environmental security, the use of chemical pesticide is more limited, thus plant protection Research emphasis is gradually transferred on natural antiviral substance by worker.Pass through the sieve to a large amount of natural products from microorganism Choosing is found and finds new antiviral substance.This is conducive to the expansion of plant virus mortifier screening spectrum, new Antiphytoviral suppression The acquisition of object processed establishes preferable basis to develop novel and multifunctional pharmaceutical grade protein.

On having reported Research foundation, we isolate fungi from the tobacco rhizosphere soil to grow fine and screen, and obtain The bacterial strain best to antiviral effect selects the mould XF6 bacterial strain in fermentation liquid containing albumen exciton, utilizes different albumen Purification technique is separated from the fermentation liquid of mould XF6 bacterial strain can cause the albumen of non-host plant tobacco allergic reaction to excite Son, studies the mechanism that the protein induced tobacco obtains system resistant, and relevant enzyme is defendd in the generation including tobacco early signal event Active variation, the generation and accumulation of defensive substance and the expression etc. with defence related resistance genes, mechanism of action becomes New albumen exciton control plant disease and the theoretical foundation for improving plant resistance to environment stress.It is expected to reducing chemical pesticide usage amount, drop It plays a significant role in the sustainable development of low Practice for Pesticide Residue in Agricultural Products and agricultural.

Summary of the invention

The object of the present invention is to provide a kind of mould albumen exciton, which is isolated and purified from Tobacco Root Border soil mould XF6 bacterial strain, is detected through SDS-PAGE, and applicant is named as EPTP, and sequence is SEQ ID NO.2 institute Show, corresponding nucleotides sequence is classified as shown in SEQ ID NO.1.

It is another object of the present invention to provide a kind of mould albumen exciton EPTP in improving disease resistance of plant Application.It is sprayed at plant leaf surface, it can induce plant to generate resistance, have favorable effect to virosis etc., especially suitable For tobacco resisting tobacco mosaic virus.

In order to achieve the above object, the present invention takes following technical measures:

A kind of mould albumen exciton EPTP, sequence are shown in SEQ ID NO.2.

A kind of mould albumen exciton EPTP can synthesize to obtain by amino acid sequence shown in SEQ ID NO.2, or utilize Sequence shown in SEQ ID NO.1, is obtained using biotechnology prokaryotic protein expression.

A kind of albumen exciton EPTP is improving the application in disease resistance of plant, and application process is:

EPTP solution (2.0-5.0mg/ml) is sprayed at plant leaf surface, plant can be induced to generate resistance, to phytopathy Viral disease has favorable effect.

Compared with prior art, the invention has the following advantages that

1. EPTP exciton prepared by the present invention can induce the expression of tobacco disease resistance related gene and the generation of defensive substance And accumulation, inhibit the proliferation of TMV, mitigate symptom, chlorophyll content is significantly higher than adjoining tree.For the new egg of development and application in the future White exciton and anti-virus formulation provide certain theory support.

2.EPTP has preferable control efficiency to TMV.Field plot experiment, 3 statistical findings are as follows: albumen excitation The preventive effect of son is followed successively by 64.69%, 61.26%, 61.28%.The preventive effect of Moroxydine Hydrochloride processing group is followed successively by 38.39%, 5.96%, 18.03%.Show that EPTP, better than Moroxydine Hydrochloride, can effectively inhibit TMV in tobacco body to the control efficiency of TMV Interior proliferation reduces disease incidence, postpones disease time.

3.EPTP is a kind of novel protein exciton, amino acid sequence difference and reported exciton.The albumen can be straight It connects and is sprayed on plant leaf blade surface, application conveniently.In addition, rich by the fermentation raw material source of the biological pesticide of albumen exciton exploitation It is rich, production cost is low, non-toxic and safe, nuisanceless.Albumen exciton is applied in behind field easily decomposition, noresidue, to environment without dirt Dye, it is comparatively safe to non-target organisms such as people and animals, with wide application and market prospects.

Detailed description of the invention

Fig. 1 is albumen exciton EPTP heat stability test.

Fig. 2 is that albumen exciton EPTP causes the anaphylactoid minimum concentration of tobacco.

Fig. 3 is that the tobacco PAL activity of EPTP processing changes map.* significant difference, P < 0.05

Fig. 4 is the Polyphenol Oxidase in Tobacco activity change of EPTP processing.* significant difference, P < 0.05

Fig. 5 is the Tobacco Peroxidase activity change of EPTP processing.

Fig. 6 is the NO changes of contents that EPTP handles tobacco cell different time.Significant very poor different, P < 0.01 *

Fig. 7 is H in EPTP DAB dyeing detection tobacco leaf₂O₂Accumulation.A control treatment blade；At B albumen exciton Manage blade

Fig. 8 is the extracellular pH variation of tobacco cell after EPTP processing.Significant very poor different, P < 0.01 *

Fig. 9 is the accumulation for the tobacco leaf callose that aniline blue detects EPTP induction.A and c is control；B and d 0.01g/ L protein EP-2 TP handles blade.

Figure 10 is the accumulation of the protein induced tobacco leaf phenolic substances of fluorescence detection EPTP.108h, A and a after processing, control Cell；B and b excites subprocessing cell

Figure 11 is the Tobacco Leaf mesophyll cell of clear water processing.A is not inoculated with TMV；B, C, D are inoculated with 12d, 21d, 28d after TMV.

Figure 12 is the Tobacco Leaf mesophyll cell after 21d.The processing of A, a clear water；B, b smear prevention processing；C, c are by spraying at treatment Reason；Prevention is handled by spraying by D, d.Ch represents chloroplaset；P represents TMV virion；S represents amylum body；Os, osmiophilic granules；CW generation Table cell wall.

Figure 13 is the relative amount for the TMV that prevention is organized by spraying.

Figure 14 is the relative amount of the TMV of 21d.

Figure 15 is that activator protein EPTP induces the anti-TMV of Nicotiana glutinosa.A, withered spot inhibiting rate；B, withered spot diameter；C, every square li The withered spot number of rice blade area.

Figure 16 is that activator protein EPTP is handled to the intracorporal SCAVENGING SYSTEM OF ACTIVATED OXYGEN key gene expression of tobacco It influences.

Figure 17 is that activator protein EPTP is handled to the influence of jasmonic-ethylene pathway related gene expression situation.

Figure 18 is influence of the activator protein EPTP processing to salicylate pathway related gene expression situation.

Figure 19 is that activator protein EPTP is handled to the influence with Resistant enzymes expression conditions.

Figure 20 is conversion daughter colony PCR verifying.M, DNA Maker；1,2,3,4,5 is transformant.

Figure 21 is that recombination pMD18-T plasmid is carrier PCR verifying.M, DNA Maker；1 is transformant 1.

Figure 22 is conversion daughter colony PCR verifying.M, 5000DNA Maker；H, pure water control；CK, zero load control；1,2,3, 4,5,6,7,8,9,10 be different transformants.

Figure 23 is the inducing expression of EPTP.M, albumen Maker；1, before control induction；2, after control induction；3, recombinant bacterium lures After leading.

Figure 24 is the SDS-PAGE of purified fusion albumen.M, albumen Maker；1, before recombinant bacterium induction；2, recombinant bacterium induction Afterwards；3, the sulphur oxidation protein of purifying；4, the fusion protein of purifying.

Figure 25 is that the allergic reaction of purifying protein detects.

Specific embodiment

Technical solution described in the embodiment of the present invention is if not otherwise specified routine techniques, agents useful for same is not as said especially It is bright, derive from commercialization channel.

Embodiment 1:

Detect the physicochemical property of albumen exciton EPTP:

(1) thermal stability is identified

Take EPTP (, 10 μ g/mls artificial synthesized according to sequence shown in SEQ ID NO.2) albumen respectively in 40 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C of incubation 5min, be immediately placed on ice, be cooled to room temperature to solution, carry out allergic reaction experiment. Detect the thermal stability of EPTP.

Experimental subjects is the cloud and mist 87 of growing way health, healthy growth and complete blade is taken, using the injection side of half leaf method Method, the leaf left side are clear water control, the right EPTP.Using the micro syringe without syringe needle in 20 μ L of each point injection EPTP or water.Blade is cut and taken a picture after 6d, observes on blade the form of allergy spot and allergy spot whether occur.If going out Existing allergy spot, then show that the activator protein can cause the allergic reaction of tobacco.

As the result is shown: such as Fig. 1, when temperature reaches 70 DEG C, EPTP albumen inactivates substantially.As it can be seen that EPTP albumen is in high temperature It is lower to be easy inactivation.

(2) albumen exciton EPTP causes the anaphylactoid minimum concentration of tobacco

The protein EP-2 TP for taking 0.5,1,2,5 μ g/ml of various concentration detects energy also according to above-mentioned Biological Activity Identification method Enough cause the anaphylactoid minimum protein concentration of tobacco.

As a result as follows: as shown in Fig. 2, the minimum concentration of the anaphylactoid protein EP-2 TP of tobacco can be caused to be 2 μ g/ml, it is low It cannot will substantially cause the allergic reaction of tobacco in this concentration, when being higher than this concentration, with the increase of concentration, allergy is anti- Should be just all the more strong, can cause typical anaphylactoid concentration is 5 μ g/ml.

Embodiment 2:

The Tobacco System Resistant reaction of EPTP induction:

The tobacco plant at 2 monthly ages is taken, the general 5-7 piece true leaf that grows is advisable, with 1ml without the syringe of syringe needle, by 400 μ l (10 μ g/ml) protein EP-2 TP solution is injected from vacuum side of blade, is control with PB buffer, and each 1 plant of processing is repeated 3 times.Processing Afterwards 24,48,72,96,120,144,168,192h takes a upper leaf for processing leaf, the change of measurement defence related enzyme activity respectively Change.

(1) phenylalanine lyase (PAL) determination of activity

PAL determination of activity is slightly changed (Hano 2008) referring to Hano literature method.The leaf tissue for taking 0.25g to handle It is added 3ml buffer (5mmol/L beta -mercaptoethanol, 1mmol/L EDTA, 1%PVP, pH 8.4), quartz sand is added in mortar Middle ice bath grinding, 4 DEG C, 8000rpm/min is centrifuged 20min, collects supernatant and detects PAL enzymatic activity.4 μ l of supernatant is taken, is added 3.9ml is measured in liquid (L-phenylalanine 20mmol/L, 0.05mmol/L borate buffer, pH 8.4), anti-at 40 DEG C after mixing 1h is answered, measures OD value in 290nm.1 unit of activity is defined as with the OD value variation 0.01 per minute of every gram of protein.

PAL activity (0.01 △ ODg^-1·min^-1)=(100 × Δ OD₂₉₀×V_T)/(FW×t×V₁)

T in formula: reaction time (min)；V_T: sample liquid total volume (ml)；V₁: amount of samples (ml) when measurement；FW: sample is fresh Weight (g).

As the result is shown: such as Fig. 3, after protein EP-2 TP induction, phenylalanine lyase (PAL) activity change is larger.EPTP 48h PAL activity dramatically increases after processing, is 1.7 times of control, is declined when to 72h, and 96h is begun to ramp up, then As the extension enzymatic activity of time declines.

(2) polyphenol oxidase (PPO) determination of activity

It is slightly changed (Ali 2006) referring to Ali literature method.The enzyme that 3ml is added in the leaf tissue for taking 0.25g to handle mentions Liquid (50mmol/L phosphate buffer, pH 7.0) is taken to be homogenized, 4 DEG C, 12000rpm/min is centrifuged 20min, and supernatant is stored in -20 DEG C, it is used for Enzyme assay.0.1ml enzyme solution is taken, 1.5ml 0.05mol/L phosphate buffer is added, reacts 2min at 30 DEG C, then 1.5ml 20mmol/L catechol is added, is measured at 420nm, is become with the OD value of every gram of protein 420nm wavelength per minute Changing 0.01 to be defined as 1 vigor is unit.

PPO activity (△ ODg^-1·min^-1)=(Δ OD₄₂₀×V_T)/(FW×t×V₁)

T in formula: reaction time (min)；V_T: sample liquid total volume (ml)；V₁: amount of samples (ml) when measurement；FW: sample is fresh Weight (g).

As the result is shown: such as Fig. 4, when EPTP handles tobacco 4d, polyphenol oxidase content control group and processing group PPO's Activity reaches highest, a higher peak value occurs, and the activity of processing group PPO is significantly higher than control group at this time, then under Drop.The POD activity of processing group is higher than control group in the most of the time section of experiment.

(3) peroxidase (POD) determination of activity

The determination of activity of POD uses o-methoxyphenol method, slightly changed (Zhang 2004).POD enzyme solution extracting method Crude enzyme liquid is diluted 10 times with 50mmol/L phosphate buffer, takes 1ml enzyme solution, 1ml 50mmol/L phosphoric acid buffer is added by same PPO Liquid adds after 1% guaiacol of 1ml shakes up, sets water-bath 5min at 30 DEG C, 1ml 3%H is then added₂O₂, under 470nm Measurement, with the OD value variation 0.01 of every milligram of protein 470 wavelength per minute for 1 unit of activity.

POD activity (△ ODg^-1·min^-1)=(Δ OD470 × VT)/(FW × t × V1)

T in formula: reaction time (min)；VT: sample liquid total volume (ml)；V1: amount of samples (ml) when measurement；FW: sample is fresh Weight (g).

As the result is shown: such as Fig. 5, activator protein EPTP can be with peroxide activator enzyme.After EPTP induction, since 2d The activity of POD is apparently higher than control group, is 1.7 times of control group, and processing group POD activity reaches maximum in 4d, is control group 1.4 times, and control group POD activity is always maintained at stabilization.

(4) EPTP induces the generation of NO

30ml tobacco suspension cell is taken, 10 μ g/ml protein EP-2 TP processing is added, then according to nitric oxide detection kit It is operated, is measured at 540nm using spectrophotometer.It is control with albumen buffer.

As the result is shown: such as Fig. 6, the tobacco cell of protein EP-2 TP processing, the yield of NO is significantly higher than in 30min-60min Control group, in protein EP-2 TP processing 60min or so, the yield of processing cell NO reaches peak, then continue at any time and It drops back to and tends to be steady, and the variation within the entirely processing time of the yield of control cell NO is little.

(5) EPTP activates the deposition of hydrogen peroxide

It is slightly changed (Hano 2008) referring to 3,3 '-diaminobenzidine (DAB) Histological stain methods of Hans.With 10 μ The protein EP-2 TP of g/ml injects tobacco leaf, after processing for 24 hours, by the blade of processing, is cleaned and is placed in plate with distilled water, taken A liquid (1mg/ml, pH 3.8) in appropriate DAB colour reagent box, is added in pipe, room temperature is kept in dark place with C liquid tune pH value to 5.8 6 hours.Then removal dye liquor is added several minutes of 95% ethyl alcohol boiling water bath, removes chlorophyll, absorbs each pipe liquid, is added anhydrous Ethyl alcohol and boiling water bath are until leaf green is sloughed completely.Blade is immersed in 70% glycerol, micro- sem observation.With albumen Buffer is control, and each processing is in triplicate.

As the result is shown: such as Fig. 7, after protein EP-2 TP handles tobacco leaf, can be observed to generate in DAB and blade and accumulation H₂O₂It reacts, the maroon spot of formation.It can be seen from the figure that hydrogen peroxide in the blade of activator protein EPTP processing It is mainly collected in nerve structure and treatment site, shows that activator protein EPTP can induce the generation of tobacco leaf active oxygen.

(6) alkalization of EPTP inducing cell expolasm

The tobacco suspension cell for taking about 0.1g/ml is transferred in new tobacco suspension cell culture medium, 25 DEG C, 150rpm/ After min is cultivated 3 days, protein EP-2 TP processing is added, makes its final concentration of 10 μ g/ml, continues to cultivate.Hereafter every 45min pH Analyzer measures the variation of the pH value of cell culture medium.It is control with albumen buffer, it is each to handle in triplicate, and the side of progress Difference analysis.

As the result is shown: such as Fig. 8, after protein EP-2 TP evoking tobacco suspension cell, substantially changeing the pH of extracellular matrix.? The extracellular pH of 90min is declined close to 6.6, alkalization degree highest in the subsequent extracellular pH of 45min after induction, then basic It is constant；Although and also being risen to pH in entire detection process is impinged upon, up to 6.3 or so, far smaller than experimental group pH.Show that activator protein EPTP can induce the alkalization of tobacco cell expolasm.

(7) EPTP induces the generation of callose

The protein EP-2 TP injection treatment tobacco leaf of 10 μ g/ml, for 24 hours after, by blade be put into buffer (1% glutaraldehyde, 5mmol/L citric acid, 90mmol/L disodium hydrogen phosphate, pH 7.4) overnight.Blade is taken out, boiling water bath in dehydrated alcohol is put into 10min takes out blade and places into 50% ethyl alcohol, is then transferred to wash buffer (67mmol/L dipotassium hydrogen phosphate, pH 12). 1h is dyed in dyeing liquor (0.1% aniline blue, 67mmol/L dipotassium hydrogen phosphate, pH 12), is taken out blade and is rushed with wash buffer It washes 1-2 times, is put into 70% glycerol, is observed with fluorescence microscope.

As the result is shown: such as Fig. 9, after carrying out callose dyeing with aniline blue, the tobacco leaf of protein EP-2 TP processing is ultraviolet Under fluorescence microscope, the fluorescence of blue can detecte, the blade of albumen buffer processing is then almost without fluorescent material Accumulation.After EPTP processing, callose is generated as plant physical defensive substance, is conducive to the ingredient for reinforcing cell wall, is formed and planted The physical mechanism of object defence.

(8) EPTP induces the generation and accumulation of phenols

Taking 300 μ l tobacco suspension cells, protein EP-2 TP, which is added, makes its final concentration of 10 μ g/ml, in 25 DEG C, 150rpm/min After being incubated for 108h, is eluted cell 1-2 times, cell is placed on after being sealed on glass slide with coverslip with PBS, it is micro- in Ultraluminescence The accumulation of phenolic substances is observed under mirror (excitation wavelength 395nm).

As the result is shown: such as Figure 10, phenolic substances is under fluorescence it can be observed that autofluorescence.It is outstanding that activator protein handles tobacco After floating cell 108h, with fluorescence microscope, the generation and accumulation that experimental group phenolic substances can be observed are apparently higher than control Group, activator protein EPTP evoking tobacco cell generates a large amount of phenolic substances, and control group generates minimal amount of phenolic substances.

Embodiment 3:

Activator protein EPTP is detected to the preventive effect of tobacco mosaic virus (TMV)

(1) indoor control tobacco mosaic virus disease is tested

The preparation of TMV virus juice: the fresh tobacco leaf with obvious virus symptoms of 1g is weighed, mortar is put into In, PBS (pH 7.0) 40mL and 2g quartz sand of 0.05M is added, after being ground into homogenate, 2 layers of filtered through gauze, obtained virus filter Liquid (30 virion/ml) can be used to frictional inoculation tobacco, and (every plant of tobacco virus inoculum concentration is with this plant of tobacco of uniform fold Top two panels young leaves is advisable), it is used for following experiment or embodiment.

The tobacco that 7-8 leaf phase growing way is consistent, healthy and strong is selected, using pot experiment, measures activator protein EPTP to tobacco The preventive effect of leaf disease viral disease.Test two kinds of modes of action of code insurance shield and treatment:

Protection group: being inoculated with TMV after application 1 time, 5 days, was then administered every 7 days 1 time, is administered that 3 times (3 times herein are not altogether Including 1 application before inoculation)；

Treatment group: being first inoculated with TMV, was then administered every 7 days 1 time, is administered 3 times altogether；

Morbidity control group: only inoculation TMV is not administered；

Super quick protein control group: application method is identical as protection group.

The above components do not use super quick albumen (Harpin) spraying (60mg/kg, every plant sprays 50ml), EPTP (10 μ g/ Ml) spraying (every plant sprays 50ml) ,+10% (V/V) lipopeptid of EPTP (10 μ g/ml) (are mentioned from bacillus amyloliquefaciens fermentation liquid Take purifying, Gaofu, 2010) spraying (every plant sprays 50ml), EPTP (10 μ g/ml)+10% lipopeptid smearing (leaf every square centimeter Piece smears 1ml)

21 days and 28 days inspection incidences after inoculation calculate disease index and control efficiency.30 plants of tobacco seedlings of every processing.System It counts the sick series of every plant of tobacco and calculates disease index and control efficiency.Disease scale standard reference literature (Chen Li and An Derong, 2004) method.

Disease index=[∑ (sick series × diseased plant number))/(highest disease grade × total strain number)] × 100

Control efficiency %=(control disease index-processing disease index)/control disease index × 100%

As the result is shown: as it can be seen from table 1 the protective effect for wherein smearing processing with activator protein EPTP is maximum, 21d and The control efficiency of 28d respectively reaches 66.7% and 39.29%, is significantly higher than the super quick albumen of comparison medicament.In addition, different application sides Formula influence EPTP preventive effect, wherein when 21d activator protein EPTP be sprayed treatment group effect it is worst, secondly for be sprayed prevention group, And the effect that EPTP+ lipopeptid is smeared is best.But the effect of protection group is best by spraying at 28d days.Analysis, protecting effect is better than treatment Effect；Scribble effect is better than spray effect.Lipopeptid is added, is conducive to improve preventive effect early period, but unobvious in later period synergy.

Protective effect for activator protein EPTP to tobacco mosaic virus disease.

Table 2 is that field trial measures activator protein EPTP to the control efficiency of tobacco mosaic virus (TMV)

Table 3 is influence of the activator protein EPTP to chlorophyll content in susceptible tobacco body.

Table 1 is protective effect of the activator protein EPTP to tobacco mosaic virus disease

(2) field control tobacco mosaic virus disease is tested

Field is administered 5 times altogether, and seedling stage application is primary, 15 days after transplanting, is respectively administered primary, EPTP within 30 days, 45 days, 60 days (10 μ g/ml) solution is sprayed the blade of whole strain tobacco (every plant sprays 50ml).Every cell plants 50 plants of clouds and mists 87, is repeated 4 times.From shifting It plants the same day and investigates a state of an illness, it is primary every investigation in 15 days.To spray clear water as blank control, spraying Moroxydine Hydrochloride is medicament Control.(tobacco mosaic disease is natural occurrence, not virus inoculation).

As the result is shown: from table 2 it can be seen that control group morbidity is the most serious, the morbidity of activator protein EPTP processing group is most light. Compared with Moroxydine Hydrochloride medicament group, the state of an illness of activator protein EPTP processing group is lighter.The preventive effect of EPTP is relatively stable, prevents three times Effect is followed successively by 64.69%, 61.26%, 61.28%, illustrates that the activator protein can prevent and treat virosis to permanent holding effect.EPTP's is anti- Effect is higher than Moroxydine Hydrochloride chemical agent, and explanation can be used using the antiviral microbial inoculum of mould activator protein EPTP instead of chemical agent In production.

Table 2 is that field trial measures activator protein EPTP to the control efficiency of tobacco mosaic virus (TMV)

(3) the variation of tobacco leaf chlorophyll content is detected

According to the experiment method of the protection group in step (1):

Start within 12 days sampling after connecing virus and then sampling in every seven days is primary, adopts four groups altogether, be respectively as follows: 1. clear water control； 2. super quick albumen (Harpin) (spraying)；3. EPTP (10 μ g/ml) (spraying)；4. EPTP (10 μ g/ml) (smearing), the spray of each group Mist amount and number are identical as in step (1), and viral inoculum concentration is identical as in step (1).

The measurement of chlorophyll content: accurately weighing 0.2g young leaflet tablet, and a small amount of CaCO is added₃And quartz sand, 5ml 95% Acetone-ethanol solution, ice bath are ground into homogenate, add 95% acetone-ethanol solution of 5ml, continue to grind, and stand 3min extremely Fragment is colourless.Filter paper filtering, filtrate is filtered into 25ml volumetric flask, with solvent washing mortar and filter paper, until colourless, constant volume To 25ml, shake up.With solvent school zero, absorbance is measured respectively at 663nm and 645nm.It is dense that chlorophyll is calculated according to the following formula out Degree:

Chlorophyll-a concentration (mg/L): C_a=12.7A₆₆₃-2.69A₆₄₅

Chlorophyll b concentration (mg/L): C_a=22.9A₆₄₅-4.68A₆₆₃

Chlorophyll total concentration (mg/L): C_a+b=C_a+C_b

It calculates and extracts leaf Determination of Chlorophyll concentration, be scaled every gram of fresh leaf chlorophyll content (mg/L), it may be assumed that

Chlorophyll concentration (mg/L) * extracting solution total volume (ml)/[sample fresh weight (g) * 1000]

As the result is shown: from table 3 it can be seen that handling position and upper blade middle period after cloud and mist 87 applies activator protein EPTP Chlorophyll contents are higher than clear water control group, and spraying smearing group becomes apparent with interlobate difference is compareed.

Table 3 is influence of the activator protein EPTP to chlorophyll content in susceptible tobacco body

Each processing group of activator protein EPTP, after being inoculated with TMV 12d, superior leaf measuring chlorophyll content the results are shown in Table 3, from As can be seen that 3 processing tobacco leaf Determination of Chlorophyll a, chlorophyll b and Chlorophyll increase compared with the control in table, 22nd day, Chlorophyll growth rate maximum in the tobacco leaf of EPTP is sprayed, is 15%.The result shows that activator protein EPTP and Super quick albumen has certain protective effect to the decline of the tobacco leaf Determination of Chlorophyll content as caused by TMV.Illustrate that albumen excites Molecular immune system in sub- EPTP energy activated plant body, improves its own immunity, passes through signaling molecule transduction regulation plant Associated metabolic process improves chlorophyll content, enhances plant photosynthesis.

(4) the research of tobacco mosaic virus (TMV) infected and organize Ultrastructural pathological change

Above-mentioned sample preparation is less than to the fritter of 1mm at diameter, sample preparation passes through transmission electron microscope observing eucaryotic cell structure.

As the result is shown: the ultra microstructure of tobacco leaf is observed under transmission electron microscope.Such as Figure 11, as a result, it has been found that, non-virus inoculation When its cellular morphology it is normal, chloroplaset marshalling and contain proper starch grain；And after being inoculated with TMV 12d, cellular morphology calibration Often, the membrane structure that chloroplaset occurs in slight enlargement cell increases, and class lysosome and particulate material increased significantly, in cell A large amount of ribosomes is dispersed with around plasma membrane.Be inoculated with 21d tobacco mesophyll cell Chloroplast metamorphosis clearly, leaf Green body expands deformation and quantity is reduced, and amylum body disappears, and the tobacco cell chloroplaset layer for being inoculated with 21d is disorganized, loose, seriously Distortion；Without amylum body and deformation is expanded in chloroplaset, is finally resulted in most of chloroplasets and is even disintegrated, cell membrane fractions disintegration, Thermophilic hungry corpusculum increases.

Tobacco is inoculated with 21d after TMV, and control group blade cell matter is messy, and film is ruptured and starts to be disintegrated, and chloroplaset is swollen Large deformation, most cells device, which disappears, to disintegrate, and a large amount of virion occurs in cytoplasm；And smear the leaf of EPTP prevention processing group Piece, mesophyll cell still keep completely, amylum body also being contained in chloroplaset, and form is normal, there is a small amount of disease into the cell Virion, and membrane body is generated, secondary thickening occurs for cell wall, and thermophilic hungry particle increases.In EPTP spraying treatment treatment group, mesophyll Cell also contains only a small amount of virion, and cell is still kept completely, and much starch grain is contained in chloroplaset, generates more thermophilic Particle is starved, cell wall thickeies.EPTP is sprayed in prophylactic treatment group, and chloroplaset form is normal and contains amylum body, is generated more Thermophilic hungry particle, cell wall thickening (Figure 12).

(5) quantitative fluorescent PCR of tobacco mosaic virus (TMV)

The synthesis of the first chain of extraction and cDNA of total serum IgE

With the upper blade of protein EP-2 TP processing tobacco leaf, required sample liquid nitrogen is taken to pulverize, every pipe packing 0.1g sample.1ml Trizol is added in every pipe, mixes rapidly, stands 5-10min at room temperature, and 12000rpm/min is centrifuged 5min, Abandon precipitating；Add 200 μ l chloroforms, concussion mixes, and 4 DEG C, 12000rpm/min is centrifuged 15min, draws upper layer, and 0.5ml isopropyl is added Alcohol mixes, -20 DEG C of standing 20min；Centrifugation, the rinsing of 75% ethyl alcohol, centrifugation removal ethyl alcohol, 5-10min dry.

With the quality of 1.2% Ago-Gel and ultraviolet absorption method detection RNA.The synthesis of first chain cDNA, primary operational Steps are as follows.

First chain cDNA reaction system:

It mixes gently, 42 DEG C of incubations 60min, 70 DEG C of heating 5min inactivate enzyme.The cDNA of synthesis is used for PCR as template Amplification.

RT-qPCR reaction system:

Amplification condition: 95 DEG C of initial denaturation 4min；It is adopted when 95 DEG C of denaturation 30s, 62 DEG C of annealing 30s, 72 DEG C of extension 30s, extension Collect fluorescence, 40 circulations；Last 72 DEG C of extensions 10min.

As the result is shown: such as Figure 13,14, spraying and smear activator protein EPTP to tobacco mosaic virus (TMV) (TMV) in tobacco body Proliferation it is inhibited, activator protein handle tobacco after, the danger of TMV can be effectively reduced with inducible system acquired resistance Evil；Its systemic acquired resistance synthesis is most strong when sampling for the first time and third time sample, and TMV significantly subtracts relative to the content of control It is few.TMV content is low more than compareing in tobacco after spraying EPTP, illustrates that activator protein EPTP evoking tobacco is obtained to virosis System resistant, to inhibit the breeding of virus indirectly.Different insecticide-applying way also influences tobacco to the resistance of TMV, prevention Smearing group TMV relative amount is minimum, prevents spraying group and takes second place, it is maximum to treat spraying group.

(6) protein EP-2 TP induction improves Nicotiana glutinosa to TMV resistance

The preparation of TMV virus juice organizes 3 young plants, 3 repetitions with example 3, growing way health, the Nicotiana glutinosa of 6-7 leaf phase.Choosing 2 blades on plant top are taken, 20 μ l of EPTP is injected.Dipped after 1d viral juice and quartz sand (1mL virus filtrate/blade, 0.1g quartz sand/blade), it is carefully and homogeneously applied on rest blade along vein.It is compared with distilled water.In inoculation disease 72h observes the form and quantity of withered spot after poison.

Withered spot inhibiting rate is calculated according to the following formula:

Withered spot area inhibiting rate=[(control group withered spot area accounts for blade gross area percentage-experimental group withered spot area and accounts for Blade gross area percentage)/control group withered spot area accounts for blade gross area percentage] × 100%

Withered spot quantity inhibiting rate=[(control group withered spot quantity-experimental group withered spot quantity)/control group withered spot quantity] × 100%

As the result is shown (after inoculation 72h observe result): such as Figure 15, protein EP-2 TP treated tobacco, withered spot area is significant Less than control, withered spot area inhibiting rate is 82%.Withered spot area is small to be mainly shown as that withered spot diameter reduces and withered spot number reduces two Aspect.It is shown in figure, withered spot diameter and withered spot number processing group are all significantly less than control.

(7) protein EP-2 TP evoking tobacco resistance related gene

A. the synthesis of the first chain of the extraction of total serum IgE and cDNA

With the upper two panels blade of 800 μ l (10 μ g/ml) protein EP-2 TP solution injection tobacco leaf, injection protein EP-2 TP is taken Neighbouring the next blade, liquid nitrogen grinding dispense 0.1g sample at powder, every pipe.RNA is extracted according to the above method, digests DNA, and carry out The reverse transcription of mRNA.

B. the design of special primer

The method that gene expression analysis uses Semiquatitative RT-PCR assay, in Gene A ctin work highly conserved in tobacco Reference.

Primer is shown in Table 4.

Table 4 is resistance related gene specific primer

C. resistant gene is expanded by RT-PCR

The the first chain cDNA product for taking above-mentioned reverse transcription, with the ddH of sterilizing₂O dilutes 50-100 times, as target gene Amplification.

Amplification condition: 94 DEG C of initial denaturation 4min；30s, 49-57 DEG C of annealing 30s of 94 DEG C of denaturation, 72 DEG C of extensions 30-45s, 35 A circulation；Last 72 DEG C of extensions 10min.PCR product detects in 1.2% Ago-Gel.

As the result is shown: exciton EPTP can induce the accumulation of plant ROS, further excite the disease resistance response of plant. After EPTP is handled tobacco 4 hours, the expression of RBHOB gene starts to significantly rise, and reached highest by 72 hours, illustrates EPTP Induction of the disease resistance response of plant.But if ROS excess accumulation, then it can make the cell membrane lipid peroxidating and membrane permeability funeral of plant It loses, so as to cause plant intracellular metabolite disorder, plant is damaged instead.If the at this time oxygen scavenging activity of energy activated plant System, so that it may so that plant improves the ability to bear of active oxygen, and alleviate ROS rapid excessive accumulation and caused by damage Wound, therefore, the up-regulated expression analysis of SCAVENGING SYSTEM OF ACTIVATED OXYGEN key gene are also a kind of performance that plant resistance to environment stress improves.APX It is a kind of SCAVENGING SYSTEM OF ACTIVATED OXYGEN key gene, it is found that the expression degree of this gene significantly improves, wherein is handled in induction APX expression quantity maximum (Figure 16) after 12h.The result shows EPTP albumen can with activated plant activity in vivo oxygen scavenging system, To increase the resistivity that tobacco infects TMV.Apoptosis is a mark of Induction of Systemic Resistance of Plant, Hin1 is the related gene of apoptosis, and in induction processing 4h, for 24 hours and after 72h, expression is all significantly raised； WIPK gene encodes wound protein kinase, and 4h starts higher expression quantity occur after exciton induction, most to 12h expression It is high.

The expression regulation of Analysis of Defence Genes Involved plays an important role in plant disease-resistant Signaling transduction networks.Induced resistance at present Signal transduction can be divided into two kinds: jasmonic/ethylene Dependent and salicylic acid Dependent, jasmonic and ethylene mainly induce alkali Property PR protein expression, such as pR3.Jasmonic/ethylene signaling pathway depends on COI1, generates defensin gene PDF.PR-3 be and this way The relevant albumen of diameter, PR-3 gene expression dose starts to increase after induction handles 4h, increases significantly, after 72h hours again after 9h Start to be declined；Although NtCOI1 gene expression after induction processing is not strong, also increased after induction processing for 24 hours Add；PDF gene transcription amount after 4h and 1d after EPTP induction all increases, and expression is continued until that observing time ties Beam；LOX is the gene that jasmonic synthesis relies on, and ERF is the related gene of ethylene pathway, and their expressions after being induced It all dramatically increases, and gene expression amount increases always, just starts to be gradually reduced to 2d.Illustrate this disease resistance response dependent on jasmine Jasmine acid, ethylene (Figure 17).

Various pathogens and chemokines all pass through salicylic acid Dependent induction plant and generate disease resistance.When exciton lures Leading increases the intracorporal SA content of plant, and the downstream signal NPR1 of bigcatkin willow acid signal is just transported from cytoplasm into nucleus, and The expression of final regulation and control and resistance related gene PR1-a/b.PR albumen is extremely important to On Induced Disease Resistance In Plants, PR1, PR2 and PR5 is salicylate pathway related gene.Wherein the expression quantity of PR-1b expression quantity after EPTP induces processing 12h just starts anxious Play increases, and later stage experssion level is also higher；And PR-1a is not so good as said gene, only just slightly increased in 2h expression； And PR-5 is dramatically increased in expression for 24 hours；Salicylate pathway marker gene NPR1 occurs after EPTP induces 2h, continues to 4th day；Induced by Salicylic Acid protein kinase (Salicylate-Induced Protein Kinase, SIPK), it is being induced 6h Transcription amount starts to increase afterwards, and keeps this amount 4d after induction；By the expression of results of these resistance related genes, say Bright EPTP can make tobacco generate the systemic resistance (Figure 18) dependent on signal pathways such as SA.

PAL enters phenylpropyl alcohol alkane approach to regulation metabolic fluxes and plays key effect, it is in addition to participating in lignin and isoflavones The generation of substance；Or the important secondary signal molecule of one kind of SA is generated, can star HR and systemic acquired resistance (SAR) expression, therefore PAL is also most important to systemic resistance of plant by quick transcriptional activation.The raising of the enzymatic activitys such as PAL It can make plant infecting from disease fungus, activity is also the important indicator (Zhang Xuekun etc. 2002) of plant resistance to environment stress.Phenylpropyl alcohol Propylhomoserin aminonialyase gene is expressed after EPTP induces 2h, and transcription amount is gradually increasing at any time, and expression quantity is always maintained at stronger water It is flat.Tobacco has been clearly enhanced to TMV when the variation tendency of different time can be seen that protein EP-2 TP in 12h from PAL activity Resistance.

Ntf4 is a kind of kinases of mitogen activated protein matter, and Npk1 is a kind of cam kinase, they are being lured It leads plant and generates and play a part of signal transmitting in resistance engineering, while adjusting the expression of related gene.PR-10 is nuclease, Act on cause of disease RNA.CRT can prevent virus in the movement of plasmodesmata, transmit with virus in iuntercellular.These genes are being lured Leading rear expression has different degrees of rising (Figure 19).

The gene expression of plant inside defense generates resistance to plant and plays central role.The inducing expression of these Analysis of Defence Genes Involved Mainly signal transduction is realized by some signaling molecules.After protein EP-2 TP handles tobacco, plant can be induced to generate system System resistance, effectively reduces the harm of tobacco mosaic virus (TMV) (TMV)；Its system resistant is comprehensive most when protein EP-2 TP evoking tobacco 1d By force.Signal pathway marker gene NPR1 and gene PR-1a/b dependent on SA are induced to express simultaneously, it was confirmed that protein EP-2 TP can Tobacco can be excited to produce the system resistant of SA signal pathway mediation.And the expression quantity liter of PDF, LOX, ERF and NtCOI1 gene Height illustrates that system resistant caused by EPTP also relies on Ja-Et approach.

Illustrate that two kinds of signaling molecule collaborations of SA and Ja-Et take part in protein EP-2 TP evoking tobacco to the resistance processes of TMV.

Embodiment 4:

The prokaryotic expression of mould albumen exciton EPTP

The genetic fragment of 537bp, full length gene 537bp are directly synthesized, sequence is the sequence shown in SEQ ID NO.1 Column 178 amino acid of coding, molecular size range 19247Da, sequence are shown in SEQ ID NO.2.

(5) target gene is transferred to bacillus coli DH 5 alpha.

Enzyme connects:

PCR product is connect with pMD18-T carrier, connects purpose according to the specification of T cloning vector pMD18-T (TaKaRa) Segment, system are as follows:

4 μ l of target DNA fragment

pMD18-T vector 1μl

Solution I 5μl

4 DEG C of connections overnight.

Conversion:

100 μ l DH5 α competent cells are taken, enzyme is added and connects 10 μ l of pMD18-T plasmid, 30min is stood in ice bath, then 42 DEG C of heat shock 90s, place into and stand 5-10min in ice bath, and 900 μ l LB liquid mediums are added, 37 DEG C, 180rpm/min, living Change 1h, takes 200 μ l to be coated on containing in Amp (100 μ g/ml) LB solid medium, 37 DEG C, cultivate 12h.

Picking monoclonal bacterial strain is added in LB liquid medium of the 1.8ml containing Amp (100 μ g/ml), 37 DEG C, 200rpm/ Min cultivates 12h, carries out thallus PCR verifying and send in AudioCodes company sequence verification positive colony.

As the result is shown: if Figure 20,21 are bacterium colony PCR, to recombinate pMD18-T plasmid as carrier PCR, and transformant is sequenced As a result show that EPTP is successfully transferred to up to 100% with target gene EPTP matching degree.

(6) target gene is transferred to e. coli bl21, carrier pET-32a.

Design of primers:

EPTP-F 5’-CGCGGATCCATGAAGTTCGTATCAGCC-3’

EPTP-R 5’-CCCAAGCTTTCAGTGTCCACCAATACTGAACC-3’

Target gene amplification, system are as follows:

Digestion target fragment and pET-32a plasmid, using Bam H I and the Hind III of TaKaRa company, and it is pure with DNA Change kit recycling, system is as follows:

T4 ligase connects digestion target gene and plasmid, system are as follows:

Conversion: method of the method for transformation referring to DH5 α.

As the result is shown: if Figure 22 is bacterium colony PCR, and 1 sequencing result of transformant is reached with target gene EPTP matching degree 100%, show that EPTP is successfully transferred to.

(7) inducing expression is transferred to the e. coli bl21 of recombinant plasmid:

A. the glycerol stock BL21 (pET-32a) (control group) and above-mentioned conversion for taking 3 μ l to save successfully contain target gene BL21 (experimental group) be inoculated in 3ml LB (containing 100 μ g/ml Amp) respectively, 37 DEG C are activated overnight.

B. by 1% inoculum concentration switching with 200ml LB (containing 100 μ g/ml Amp), 37 DEG C, 180rpm/min is cultivated 3h。

C. the IPTG that final concentration of 1mM is added after culture 3h is induced, and 37 DEG C, after 180rpm/min cultivates 5h, 8000rpm/ Min is centrifuged 5min, collects thallus.

D. thallus is resuspended with PBS buffer solution, 8000rpm/min is centrifuged 5min, collects thallus.

E. ultrasonic disruption cell: thallus is resuspended with 20ml PBS buffer solution, the PMSF of final concentration of 1mM is added, super Ice bath is crushed 1h (interval 200W, 5s, 5s) on sonication instrument.

SDS-PAGE detection:

Before taking control to induce (before adding IPTG), 5h after induction, the bacterium solution 1.5ml of 5h after experiment induction, thalline were collected by centrifugation Afterwards, 50 μ l PBS buffer solution are added to be resuspended, add 10 μ l sample-loading buffers, after boiling 5-10min, centrifuging and taking supernatant, loading, The expression of 12%SDS-PAGE electrophoresis detection recombinant protein.

As the result is shown: as Figure 23 obviously has more an egg after control induction compared with before control induction on 18.4KDa Informal voucher band, for the sulphur oxidation protein for the pET-32a plasmid expression being transferred to；One is obviously had more on 35KDa after recombinant bacterium induction Protein band, for the fusion protein of the sulphur oxidation protein and EPTP albumen of the recombination pET-32a plasmid expression being transferred to.

(8) Ni-NTA isolates and purifies fusion protein

A. the BL21 bacterial strain for being transferred to recombinant plasmid, sonicated cells, 8000rpm/min centrifugation are induced according to the above method 30min.Supernatant is precipitated and carries out SDS-PAGE detection, the results show that fusion protein is present in precipitating.With the urine of the 8M of 20ml Element denaturation 12h, makes it dissolve, 8000rpm/min is centrifuged 10min and collects supernatant.

B. after filler dress column, distilled water (3-5 times of bed volume) cleans ethyl alcohol, slow with 5-10 times of bed volume Ni column equilibration Fliud flushing (the Tris-HCl of 100ml, 0.2M；100ml glycerol；29.22g NaCl constant volume is washed to 1L), then with the urea containing 8M Ni column balance buffering liquid balances pillar, and computer Ultraviolet Detector returns to zero under 280nm ultraviolet light.

C. loading: 20ml supernatant whole loading.

D. it after completion of the sample, is eluted with the Ni column balance buffering liquid of the urea containing 8M, during which receipts minimum to being dropped to through peak Collection penetrates peak.

E. memebrane protein (10-15ml) is eluted with 1%DOC, collects sample.

F. foreign protein is eluted with the 8M urea Ni column balance buffering liquid of the imidazoles containing 20mM, until crest line is steady.

G. fusion protein is eluted with the 8M urea Ni column balance buffering liquid of the imidazoles containing 200mM, collects sample.

The recycling of h.Ni-NTA column.

The renaturation of fusion protein

It is successively dialysed with the PBS buffer solution containing 6M, 4M, 2M, 0M urea, each 12h.

Ni-NTA isolates and purifies sulphur oxidation protein

It is similar that purification process and Ni-NTA isolate and purify fusion protein method, since sulphur oxidation protein is after clasmatosis In supernatant, therefore need not be urea-denatured with 8M again, buffer used uses Ni column balance buffering liquid to dissolve.

SDS-PAGE detects purifying protein.Anaphylaxis test experience is carried out with fusion protein and sulphur oxidation protein after renaturation.

As the result is shown: such as Figure 24, purifying obtains purer albumen.Such as Figure 25, anaphylaxis detection display fusion protein still has Active, sulphur oxidation protein is unable to evoking tobacco and generates allergic reaction.

SEQUENCE LISTING

<110>Hua Zhong Agriculture University

<120>a kind of mould albumen exciton EPTP and its application in raising disease resistance of plant

<130>a kind of mould albumen exciton EPTP and its application in raising disease resistance of plant

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 537

<212> DNA

<213>artificial sequence

<400> 1

atgaagttcg tatcagccgc tgccctgctt ctcgccgctc cgctcgtcgc ggcggcgtcc 60

atccccagca tcttcgaccc gacgcaggct cccatgagtg cggccaacag cgtgcgaaac 120

aacccggtcg agggtgacaa tcctttggaa ttctgtgaac ctcctacgtc tcatcttctc 180

accatcgact ccgtcgactt gtctcccaac ccccccaaag ctggccagac cctcactatc 240

accgcctcgg gcgtcttcca cgaacgcatt acctccggcg ccacggtaaa cattgttgtc 300

aaatatggtc tcatcacctt gatcaggcag actatggatc tctgcgaaca gattgagaac 360

gtcgacttga aatgccctct cgagaaggga agaatgacca tgaccaagca ggtggacctg 420

ccaagccaaa ttcctcccgg cacctattcc gtccttgctg atgtctacac cgaggaccac 480

aagaaggtca cctgcctcat ggctaatggc atccggttca gtattggtgg acactga 537

<210> 2

<211> 178

<212> PRT

<213>artificial sequence

<400> 2

Met Lys Phe Val Ser Ala Ala Ala Leu Leu Leu Ala Ala Pro Leu Val

1 5 10 15

Ala Ala Ala Ser Ile Pro Ser Ile Phe Asp Pro Thr Gln Ala Pro Met

20 25 30

Ser Ala Ala Asn Ser Val Arg Asn Asn Pro Val Glu Gly Asp Asn Pro

35 40 45

Leu Glu Phe Cys Glu Pro Pro Thr Ser His Leu Leu Thr Ile Asp Ser

50 55 60

Val Asp Leu Ser Pro Asn Pro Pro Lys Ala Gly Gln Thr Leu Thr Ile

65 70 75 80

Thr Ala Ser Gly Val Phe His Glu Arg Ile Thr Ser Gly Ala Thr Val

85 90 95

Asn Ile Val Val Lys Tyr Gly Leu Ile Thr Leu Ile Arg Gln Thr Met

100 105 110

Asp Leu Cys Glu Gln Ile Glu Asn Val Asp Leu Lys Cys Pro Leu Glu

115 120 125

Lys Gly Arg Met Thr Met Thr Lys Gln Val Asp Leu Pro Ser Gln Ile

130 135 140

Pro Pro Gly Thr Tyr Ser Val Leu Ala Asp Val Tyr Thr Glu Asp His

145 150 155 160

Lys Lys Val Thr Cys Leu Met Ala Asn Gly Ile Arg Phe Ser Ile Gly

165 170 175

Gly His

Claims (1)

1. Albumen shown in 1.SEQ ID NO.2 encodes protein nucleotide sequence shown in SEQ ID NO.2 in raising tobacco plant pair Application in tobacco mosaic virus (TMV) disease resistance.